Ventricular arrhythmias have diverse and complex etiologies that include excessive stimulation of multiple neurohumoral pathological pathways, suggesting that no single upstream blocker will be sufficiently efficacious. Ca[2+]/calmodulin-dependent protein kinase II (CaMKII) is a common downstream mediator of these neurohumoral pathways and its hyperactivity contributes to the mechanisms of ventricular arrhythmia. We propose that the kinase is a novel therapeutic target, an idea supported by the demonstration that pharmacological and genetic reduction of CaMKII activity decreases arrhythmia in animal models and in human cardiomyocytes from patients with arrhythmia and elevated CaMKII. We have developed small molecule inhibitors of CaMKII and propose to increase potency and selectivity of the lead compound then test the best compounds for inhibition of arrhythmia in a robust mouse model. Guided by insights we developed from docking inhibitors to its active site in our new crystal structures we propose a set of compounds designed for lead optimization. Our goal is to improve potency 10-fold and reach IC50s below 15 nM. The preclinical proof-of concept study will test the in vivo efficacy of the top inhibitors using a calcineurin over expressing mouse model that is relevant to larger animal models, and likely to diseased human myocardium as well. The mice have severe heart failure and high levels of ambient arrhythmias resulting from increased CaMKII expression and our milestone is to show efficacy with at least one CaMKII inhibitor. If successful we will be positioned to extend the preclinical development by optimizing the inhibitors for ADME/Tox and other drug-like properties, testing their efficacy in other animal models, and perform IND enabling studies for an IND application aimed at arrhythmia in heart failure. PUBLIC HEALTH RELEVANCE: Sudden cardiac death is a major public health problem, which is estimated to kill 500,000 Americans each year. Most sudden cardiac death is due to rapid ventricular arrhythmias and patients with heart failure are at highest risk. Evidence now supports that pharmacological targeting of intracellular signaling, in particular of Ca2+/calmodulin-dependent protein kinase II, a sensor of dysregulated calcium homeostasis, will inhibit arrhythmia in heart failure, and it is thus a novel target in arrhythmia. We propose to modify a small molecule inhibitor we developed for this protein kinase in ways that increase its potency, analyze the new inhibitors biochemically to ensure they have the desired properties, and then test for inhibition of arrhythmia in a mouse model as a proof-of-concept.